Incubation of interleukin-2 (IL-2) with human peripheral blood mononuclear cells (PBMC) or mouse splenocytes induces a population of highly tumoricidal cells. This phenomenon has been referred to as lymphokine-activated killer (LAK) cell activity. The precursor of the LAK effector cells may be heterogeneous, but most of the activity apparently originates from large granular lymphocytes (LGL) which comprise about 5% of peripheral blood lymphocytes (PBL) and which have natural killer (NK) cell activity.
Adoptive transfer of LAK cells to tumor-bearing mice with simultaneous administration of IL-2, has resulted in reduction in tumor burden in several animal models. From these results clinical trials were developed utilizing LAK cells alone. IL-2 alone, and finally an intensive treatment program utilizing both agents in patients with advanced solid tumors (Rosenberg et al., (1987) N. Engl. J. Med. 316:889-897: U.S. Pat. No. 4,690.915 issued to Rosenberg). The toxicity of this combination regimen was considerable despite the fact that Rosenberg was able to deliver in man only 1 to 10% of the equivalent dose compared to the effective murine doses of LAK cells and IL-2 (based on weight). Nevertheless, a significant number of partial responses were seen and further trials of the combination of IL-2 and LAK cells are underway.
Central to the problem of the utilization of LAK cells in man is the complexity of their generation (also referred to herein as induction or activation): patients are leukapheresed, the leukapheresis product is separated by Ficoll-Hypaque gradients, and the resultant mononuclear cells are then cultured in the presence of IL-2 at cell densities of 1 to 3.times.10.sup.6 cells per mL for 3 to 5 days. Thus, the final culture volumes in roller bottles can reach 40 L. Various changes and improvements have been made in this procedure. For example, European patent application 87107755.8, published Dec. 2, 1987, and co-assigned, allowed U.S. patent application 07/038361, filed Apr. 20, 1987, now U.S. Pat. No. 4,849,329 disclose that depletion of monocytes by exposure of mononuclear cells to phenylalanine methyl ester (PME) allows LAK cell induction at cell culture densities about 10-fold greater than the currently utilized LAK cell induction concentrations. European patent application 88101138.1, published Aug. 31, 1988 and pending, co-assigned U.S. patent application 07/008273, filed Jan. 29, 1987. disclose that LAK cell induction can be carried out in bags made of organic polymeric, oxygen permeable film. European patent application 88106566.8 published Nov. 9, 1988, and co-assigned U.S. Pat. No. 4,808,151, issued February 28, 1989, disclose that the leukapheresis product need not be separated on a Ficoll-Hypaque gradient prior to use in the activation process.
Incubating mixtures of human white blood cells collected from the peripheral blood by venipuncture or leukapheresis with esters of various amino acids leads to the transient or permanent loss of functional natural killer (NK) cells (Thiele et al. (1985) Proc. Natl. Acad. Sci. USA 82:2468-2472: U.S. Pat. No. 4,752,602 issued to Lipsky and Theile). With the methyl ester of leucine (LME), a dipeptide leucyleucine methyl ester is formed in cells exposed to LME, which is toxic to the NK cells (Thiele et al. (1985) Proc. Acad. Sci. USA 82:2468-2472: U.S. Pat. No. 4,752,602). The use of PME and other lower alkyl amino acid esters, including the esters of alanine, aspartic acid, cysteine, glutamic acid, glutamine, phenylalanine, proline, tyrosine, tryptophan, and valine, or a mixture of any of the foregoing, in a process to prepare LAK cells is disclosed in European patent application 87107755.8, published Dec. 2, 1987, and allowed U.S. application 07/038361. filed Apr. 20, 1987, now U.S. Pat. No. 4,849,329. A problem still exists with the processes described in the art due to the complexity and high volume of the induction or activation systems needed for human LAK cell immunotherapy. The present application describes a process for LAK cell activation at high cell density therefore rendering murine equivalent doses of LAK cell therapy in humans as a feasible alternative.